FIG 2.

Replacement of endogenous counterselectable markers fcyB, fcyA, and uprt by DNAs of interest (DOIs). (a) Scheme of the generation of knock-in constructs. 5′ and 3′ nontranslated regions (NTRs) (PCR1) of the respective loci as well as the DOIs (PCR2; GFP or lacZ reporter cassette) are amplified from genomic DNA (gDNA) and plasmid DNA, respectively. Both NTRs and DOIs contain overlapping DNA (gray line) for subsequent connection via fusion PCR, yielding the knock-in constructs. (b) Double crossover homologous recombination-based replacement of fcyB, fcyA, or uprt by DOIs. Transformation selection was conducted using 5FC (fcyB and fcyA locus) or 5FU (uprt locus). (c) Visualization of GFP as well as LacZ expression in the corresponding knock-in strains after incubation on solid AMM for 48 h at 37°C.